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Chikungunya ELISA Protocol
酶联免疫吸附法(ELISA)测定基孔肯雅病毒   

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Abstract

This protocol will result in the accurate qualitative measurement of anti-Chikungunya virus antibody (Ab) from infected mouse tissue or serum. This assay was developed by Dr. Caitlin Briggs, Arbovax, Inc. Chikungunya is a BL3 agent and should be handled in a biosafety level 3 laboratory under BL3 conditions. This protocol was used in the publication “Chikungunya virus host range E2 transmembrane deletion mutants induce protective immunity against challenge in C57BL/6J mice” (Piper et al., 2013).

Materials and Reagents

  1. Baby hamster kidney (BHK) cells (ATCC, catalog number: CCL-10 )
  2. Chikungunya Virus (CHIKV)
  3. Potassium tartrate
  4. 15% potassium tartrate in Dulbecco’s phosphate buffered saline (PBS-D) (Sigma-Aldrich)
  5. 35% potassium tartrate in PBS-D (Sigma-Aldrich)
  6. Micro BCA protein assay kit (Pierce, catalog number: 23235 )
  7. Goat anti-mouse IgG HRP (Sigma-Aldrich, catalog number: A5278 )
  8. ABTS Peroxidase Substrate System (KPL, Kirkegaard & Perry Laboratories, catalog number: 50-62-00 )
  9. Cavicide disinfectant
  10. Bleach
  11. MEM (Life Technologies, catalog number: 11095 )
  12. Sodium carbonate (Sigma-Aldrich)
  13. Sodium bicarbonate (Sigma-Aldrich)
  14. Tween-20 (Fisher Scientific)
  15. Heat Inactivated Fetal Calf Serum (FCS)
  16. Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich)
  17. Gentamicin sulfate (Life Technologies, catalog number: 15710 )
  18. Potassium chloride (Sigma-Aldrich)
  19. Potassium phosphate (monobasic) (Sigma-Aldrich)
  20. Sodium chloride (Sigma-Aldrich)
  21. Sodium phosphate (dibasic) (Sigma-Aldrich)
  22. Completed 1x MEM (see Recipes)
  23. PBS-D (see Recipes)
  24. Wash Buffer (see Recipes)
  25. Blocking Buffer (see Recipes)
  26. Carbonate Buffer (pH 9.5) (see Recipes)

Equipment

  1. Laminar flow biosafety cabinet
  2. 37 °C incubator with 5% CO2
  3. 96-well plate reader (405 nm absorbance filter)
  4. 96-well Nunc MaxiSorp plate (Thermo Scientific, catalog number: 44-2404 -2)
  5. Microscope
  6. T-75 vented tissue culture flasks (Corning, catalog number: 430641 )
  7. Serological pipettes
  8. Pipet-aid
  9. 50 ml conical tubes
  10. Test tube rack
  11. Benchtop centrifuge with removable buckets with gaskets
  12. Weigh scale with 0.01 g sensitivity
  13. Ultra Centrifuge
  14. SW28 rotor and 38 ml and 17 ml buckets
  15. Beckman Ultra Clear centrifuge tubes (25 x 89 mm) (Beckman Coulter, catalog number: 344058 )
  16. Seton centrifuge tubes (16 x 102 mm) (Seton Scientific, catalog number: 7076 )
  17. Small hand held light/flash light
  18. Parafilm

Procedure

All of procedures A and B are performed at the BL3 level unless otherwise specified. Therefore every step involving live intact virus in unsealed containers must be completed inside a laminar flow biosafety cabinet, and the following preparative protocols should be observed before and after work is performed.

  1. Turn on UV light in hood for 5 min prior to beginning experiment.
  2. Turn off UV light and clean the area under the hood with Cavicide disinfectant.
  3. Remove all materials from the hood not needed for this experiment. Obtain clean waste receptacle containing enough bleach so that a full receptacle will have a final bleach concentration of 10%.
  4. Clean all materials going under the hood with Cavicide disinfectant prior to removal from the hood.

  1. CHIKV Infection and Purification: see also Reference 2 Sindbis virus purification
    1. Place media in 37 °C water bath to warm.
    2. Remove T-75 vented flasks containing BHK cells passaged 24 h prior to infection from 37 °C incubator with 5% CO2.
    3. Observe cell media for changes in color and clarity.
    4. Visualize cells under microscope at 20x maginification to check monolayer for irregularity, and confluence. Cells need to be 95%-100% confluent prior to infection.
    5. Pour media off flasks, and infect BHK cells with a multiplicity of infection (MOI) of 10 plaque forming units (pfu) per cell of CHIKV in 1 ml of completed 1x MEM (for infection follow Sindbis virus instructions in Hernandez et al., 2005).
    6. Rock flasks with virus at 25 °C for one hour.
    7. Add another 4 ml media to each infected flask.
    8. Place infected flasks in incubator set at 37 °C for one day.
    9. After 24 h, remove infected flasks from incubator.
    10. Harvest virus: Place flasks in hood and
      1. Transfer media from flasks into 50 ml conicals.
      2. Place conicals in gasketed buckets to spin, while still working inside the hood.
      3. Move sealed buckets to centrifuge, and spin cell media 10 min at 1,000 x g in bench top centrifuge to pellet cell debris.
      4. Inside the hood, transfer media to new conical tubes.
    11. While spinning cell media, prepare gradients inside the hood.
      1. Poured gradients: initial potassium tartrate step gradient. This gradient is formed by carefully layering 12 ml of 15% potassium tartrate onto a layer of 6 ml of 35% potassium tartrate in an ultraclear Beckman ultracentrifuge tube for SW28 rotor.
      2. Using a permanent marker, mark the interface of the two solutions on the outside of the tube. This will be approximately where your virus bands appear.
    12. Add 20 ml of harvested virus infected cell media to ultracentrifuge tube containing gradient gently, do not mix gradient. Must be an overlay.
    13. Place gradient tubes in Beckman rotor buckets.
    14. Balance buckets on a scale, using left over virus infected cell media to balance. Final weights must be within 0.03 grams of each other. Buckets containing virus must be sealed and cleaned with disinfectant prior to removal from the hood.
    15. Add buckets to rotor, and spin at 24,000 rpm, 4 °C overnight.
    16. Stop the spin, and place centrifuge buckets inside the hood.


      Figure 1.

      Shown (Figure 1) is the band of virus after the first spin. Note that the band is seen at the marked interface. The second gradient will be similar with a tighter band as seen below (Figure 2). There will be loss of virus however a second purification is necessary to remove most of the remaining contaminants.


      Figure 2.

    17. Set up test tube holder and a hand held light inside the hood.
    18. Place ultraclear tube in test tube rack.
    19. Using the hand held light, visualize your bands, which will be located near the interface of your gradient.
    20. Using a 25 ml pipet pull off top of gradient including all the completed medium.
    21. Using a 2 ml serological pipet, collect the virus band from the gradient, using the light to monitor the band.
    22. Dilute virus from the first gradient at least 1:2 with 1x PBS-D (Hernandez and Brown, 2010; Hernandez et al., 2005; Hernandez et al., 2010), for a total of 8 ml of virus.
    23. Set up new gradients
      1. To a 17 ml Seton ultracentrifuge tube add 3 ml of 35% potassium tartrate, then overlay with 6 ml of 15% tartrate.
      2. Gently overlay with diluted virus from step 22 above, avoid mixing the gradient.
    24. Repeat steps A13-14 above for these gradients.
    25. Spin tubes at 26,000 rpm for 4 h at 4 °C.
    26. Repeat steps A16-21, adjusting pipette size accordingly. Store collected live virus bands at 4 °C up to 2 weeks.

  2. ELISAQuantify protein in live purified virus using a micro BCA protein assay kit
    1. The night before the assay, coat a 96-well NuncMaxiSorp plate by adding 100 ng/well of purified virus in 100 μl/well carbonate buffer. Seal plate with parafilm, and incubate overnight at 4 °C.
    2. The next morning, remove carbonate buffer + virus from plate by pipetting to avoid any additional risk of forming aerosols, and do not wash.
    3. Block plate for 2 h with 100 μl/well blocking solution.
    4. Pipette off blocking buffer from plate, and do not wash.
    5. Dilute the mouse serum samples to be tested 1:100 in wash buffer, and add 100 μl diluted sample to each well of the plate (may need to try multiple dilutions to see which works best for your samples). Incubate for 1.5 h at 4 °C.
    6. Pipette off diluted serum samples, and wash plate 4x with 300 μl/well of wash buffer, removing old wash buffer before starting a new wash.
    7. Add 100 μl of goat anti-mouse IgG HRP at a dilution of 1:2,000 in wash buffer. Incubate 1.5 h at 4 °C.
    8. Pipette off secondary antibody, and wash plate 4x with 300 μl/well of wash buffer. Do one final wash with 300 μl/well of wash buffer, sealing plate with parafilm and inverting plate prior to removing wash buffer.
    9. Combine equal amounts solution A and B from the ABTS Peroxidase Substrate System (KPL) in a conical tube, and add 100 μl/well to the ELISA plate. Incubate 15 min at room temperature.
    10. Stop reaction with 100 μl of 1% SDS (SDS will inactivate any live CHIKV still present in the plate, allowing for removal of the plate from BL3 conditions after this step). Read plate at 405 nm.
    11. Positive ELISA results are determined by significantly higher OD values when comparing samples from animals that should produce an immune response to those of mock animals. Statistical analysis should be performed to determine significance.

Recipes

  1. Complete MEM
    MEM 
    10% FCS
    0.1% gentamicin sulfate
  2. Dulbecco’s Phosphate Buffered Saline (PBS-D) (Hernandez and Brown, 2010; Hernandez et al., 2005; Hernandez et al., 2010)
    0.2 g potassium chloride
    0.2 g potassium phosphate, monobasic
    8.0 g sodium chloride
    1.13 g sodium phosphate, dibasic
    Deionized water to 1 L
    Autoclave 20 min to sterilize
  3. Wash buffer
    PBS-D
    0.02% Tween-20
  4. Blocking buffer
    Wash buffer
    10% FCS or 1x Sigma Block
  5. Carbonate buffer (pH 9.5)
    3.39 g Sodium Carbonate
    5.70 g Sodium Bicarbonate
    Deionized water to 1 L
    You should not need to adjust pH.

Acknowledgments

The protocol described here was used in the publication Piper et al. (2013).

References

  1. Hernandez, R. and Brown, D. T. (2010). Growth and maintenance of baby hamster kidney (BHK) cells. Curr Protoc Microbiol Chapter 4: Appendix 4H.
  2. Hernandez, R., Sinodis, C. and Brown, D. T. (2005). Sindbis virus: propagation, quantification, and storage. Curr Protoc Microbiol Chapter 15: Unit 15B 11.
  3. Piper, A., Ribeiro, M., Smith, K. M., Briggs, C. M., Huitt, E., Nanda, K., Spears, C. J., Quiles, M., Cullen, J., Thomas, M. E., Brown, D. T. and Hernandez, R. (2013). Chikungunya virus host range E2 transmembrane deletion mutants induce protective immunity against challenge in C57BL/6J mice. J Virol 87(12): 6748-6757.

简介

该协议将导致从受感染的小鼠组织或血清中准确定性测量抗基孔肯雅病毒抗体(Ab)。 该测定由Dr.Citlin Briggs,Arbovax,Inc。开发。Chikungunya是BL3剂,并且应在BL3条件下在生物安全级3实验室中处理。 该方案用于出版物"Chikungunya病毒宿主范围E2跨膜缺失突变体诱导针对C57BL/6J小鼠中的攻击的保护性免疫"(Piper等人,2013)。

材料和试剂

  1. 幼仓鼠肾(BHK)细胞(ATCC,目录号:CCL-10)
  2. 基孔肯雅病毒(CHIKV)
  3. 酒石酸钾
  4. 15%酒石酸钾在Dulbecco's磷酸盐缓冲盐水(PBS-D)(Sigma-Aldrich)中
  5. 35%酒石酸钾的PBS-D(Sigma-Aldrich)
  6. Micro BCA蛋白测定试剂盒(Pierce,目录号:23235)
  7. 山羊抗小鼠IgG HRP(Sigma-Aldrich,目录号:A5278)
  8. ABTS过氧化物酶底物系统(KPL,Kirkegaard& Perry Laboratories,目录号:50-62-00)
  9. 杀菌消毒剂
  10. 漂白
  11. MEM(Life Technologies,目录号:11095)
  12. 碳酸钠(Sigma-Aldrich)
  13. 碳酸氢钠(Sigma-Aldrich)
  14. 吐温-20(Fisher Scientific)
  15. 热灭活胎牛血清(FCS)
  16. 十二烷基硫酸钠(SDS)(Sigma-Aldrich)
  17. 庆大霉素硫酸盐(Life Technologies,目录号:15710)
  18. 氯化钾(Sigma-Aldrich)
  19. 磷酸钾(一元碱)(Sigma-Aldrich)
  20. 氯化钠(Sigma-Aldrich)
  21. 磷酸钠(二价)(Sigma-Aldrich)
  22. 已完成1x MEM(请参阅配方)
  23. PBS-D(参见配方)
  24. 洗涤缓冲液(参见配方)
  25. 阻塞缓冲区(参见配方)
  26. 碳酸盐缓冲液(pH 9.5)(参见配方)

设备

  1. 层流生物安全柜
  2. 37℃的具有5%CO 2的培养箱中
  3. 96孔板读数器(405nm吸光度滤光片)
  4. 96孔Nunc MaxiSorp板(Thermo Scientific,目录号:44-2404-2)
  5. 显微镜
  6. T-75通气组织培养瓶(Corning,目录号:430641)
  7. 血清移液器
  8. 助洗剂
  9. 50ml锥形管
  10. 试管架
  11. 带有垫圈的可拆卸铲斗的台式离心机
  12. 称量秤具有0.01 g灵敏度
  13. 超离心机
  14. SW28转子和38毫升和17毫升水桶
  15. Beckman Ultra Clear离心管(25×89mm)(Beckman Coulter,目录号:344058)
  16. Seton离心管(16×102mm)(Seton Scientific,目录号:7076)
  17. 小手持灯/闪光灯
  18. parafilm

程序

除非另有说明,否则所有过程A和B都在BL3级别执行。 因此,在未密封的容器中涉及活的完整病毒的每个步骤必须在层流生物安全柜内完成,并且在进行工作之前和之后应当观察以下制备方案。

  1. 在开始实验之前,在罩中打开紫外线5分钟
  2. 关闭紫外线灯,并用Cavicide消毒剂清洁罩下的区域。
  3. 从机罩中取出本实验不需要的所有材料。 获得含有足够漂白剂的清洁废物容器,使完全容器的最终漂白浓度为10%
  4. 在除去罩之前,用Cavicide消毒剂清洁罩下的所有材料
  1. CHIKV感染和纯化:也参见参考文献2 Sindbis病毒纯化
    1. 将培养基置于37℃水浴中温热
    2. 在感染之前,从含有5%CO 2的37℃培养箱中移除含有BHK细胞的T-75通气烧瓶24小时。
    3. 观察细胞培养基的颜色和透明度变化。
    4. 可视化细胞在显微镜下20x maginification检查单层不规则和汇合。 感染前细胞需要95%-100%汇合
    5. 将培养基从烧瓶中倒出,并用在1ml完全的1×MEM中的每个细胞的CHIKV的10噬菌斑形成单位(pfu)的感染复数(MOI)感染BHK细胞(感染遵循Sindbis病毒说明书,在Hernandez等人 。,2005)。
    6. 将具有病毒的岩石烧瓶在25℃下保持1小时
    7. 向每个被感染的瓶中再加入4ml培养基
    8. 将感染的烧瓶置于37℃的培养箱中1天
    9. 24小时后,从培养箱中取出感染的培养瓶
    10. 收获病毒:将烧瓶放在通风橱中,
      1. 将培养基从烧瓶转移到50 ml锥形瓶中
      2. 将圆锥体放在带衬垫的桶中旋转,同时仍然在罩内工作
      3. 将密封桶移至离心机,在台式离心机中以1000 x g旋转细胞培养基10分钟,以沉淀细胞碎片。
      4. 在罩内,将介质转移到新的锥形管
    11. 同时旋转细胞培养基,在罩内准备梯度。
      1. 倾斜梯度:初始酒石酸钾梯度梯度。 该梯度通过在用于SW28转子的超级Beckman超速离心管中小心地将12ml 15%酒石酸钾层合到6ml 35%酒石酸钾层上形成。
      2. 使用永久标记,在管外侧标记两种溶液的界面。 这大约是您的病毒频带出现的位置。
    12. 加入20毫升收获的病毒感染的细胞培养基到含有梯度的超速离心管轻轻,不要混合梯度。 必须是重叠式广告。
    13. 将梯度管放置在Beckman转子轮叶中
    14. 在秤上平衡桶,使用剩余的病毒感染的细胞培养基来平衡。 最终重量必须在0.03克之内。 含有病毒的桶必须密封,并在从通风橱中取出前用消毒剂清洁
    15. 向转子中加入桶,在24,000 rpm,4℃下旋转过夜
    16. 停止旋转,并将离心机桶放置在护罩内。


      图1。

      显示(图1)是第一次旋转后的病毒带。 注意,在标记的接口处看到频带。 第二梯度将类似于更紧密的带,如下面所示(图2)。 将会有病毒的丢失,但是第二次净化是必要的,以去除大多数剩余的污染物

      图2。

    17. 在护罩内设置试管支架和手持灯。
    18. 将超声波管放在试管架上。
    19. 使用手持灯,可视化你的乐队,这将位于渐变的界面附近
    20. 使用25ml移液管从包括所有完成的培养基的梯度顶部拉出
    21. 使用2ml血清移液管,从梯度收集病毒带,使用光监测带
    22. 用1×PBS-D从第一梯度稀释病毒至少1:2(Hernandez和Brown,2010; Hernandez等人,2005; Hernandez等人,2010 ),总共8 ml的病毒
    23. 设置新渐变
      1. 向17ml Seton超速离心管中加入3ml 35%酒石酸钾,然后用6ml 15%酒石酸盐覆盖。
      2. 轻轻地覆盖上面步骤22中稀释的病毒,避免混合梯度
    24. 对这些渐变重复上述步骤A13-14。
    25. 在4℃下以26,000rpm离心4小时
    26. 重复步骤A16-21,相应地调整移液器大小。 在4°C存储收集的活病毒带,最多2周。

  2. ELISA使用micro BCA蛋白测定试剂盒测定活的纯化病毒中的蛋白质
    1. 在测定前一天晚上,通过在100μl/孔碳酸盐缓冲液中加入100ng /孔的纯化病毒来包被96孔NuncMaxiSorp板。 密封板用石蜡膜,并在4℃下孵育过夜
    2. 第二天早晨,通过吸取从板中移除碳酸盐缓冲液+病毒,以避免任何额外的风险形成气溶胶,不要洗。
    3. 用100μl/孔封闭溶液封闭平板2小时
    4. 从板上吸取封闭缓冲液,不要洗涤
    5. 稀释待测试的小鼠血清样品1:100在洗涤缓冲液,并添加100微升稀释的样品到板的每个孔(可能需要尝试多个稀释,看哪个最适合您的样品)。在4℃下孵育1.5小时。
    6. 吸取稀释的血清样品,用300μl/孔的洗涤缓冲液洗涤板4次,在开始新的洗涤之前除去旧的洗涤缓冲液。
    7. 在洗涤缓冲液中以1:2000的稀释度加入100μl山羊抗小鼠IgG HRP。在4℃孵育1.5小时
    8. 吸取第二抗体,并用300μl/孔的洗涤缓冲液洗板4次。最后用300μl/孔的洗涤缓冲液进行最后一次洗涤,用封口膜封闭平板,倒置平板,然后取出洗涤缓冲液。
    9. 在锥形管中混合来自ABTS过氧化物酶底物系统(KPL)的等量溶液A和B,并向ELISA板中加入100μl/孔。在室温下孵育15分钟。
    10. 停止反应与100μl的1%SDS(SDS将灭活仍然存在于板中的任何活的CHIKV,允许在该步骤后从BL3条件中移除板)。 在405 nm下读板。
    11. 当比较应当产生与模拟动物的免疫应答的动物的样品时,通过显着更高的OD值测定阳性ELISA结果。 应进行统计分析以确定重要性。

食谱

  1. 完成MEM
    MEM 
    10%FCS
    0.1%庆大霉素硫酸盐
  2. Dulbecco's磷酸盐缓冲盐水(PBS-D)(Hernandez和Brown,2010; Hernandez等人,2005; Hernandez等人,2010)
    0.2克氯化钾
    0.2克磷酸二氢钾 8.0克氯化钠
    1.13g磷酸二氢钠 去离子水至1 L
    高压灭菌20分钟灭菌
  3. 洗涤缓冲液
    PBS-D
    0.02%Tween-20
  4. 阻塞缓冲区
    洗涤缓冲液
    10%FCS或1x Sigma块
  5. 碳酸盐缓冲液(pH 9.5)
    3.39克碳酸钠
    5.70克碳酸氢钠 去离子水至1 L
    您不需要调整pH值。

致谢

这里描述的方案用于出版物Piper等人(2013)。

参考文献

  1. Hernandez,R。和Brown,D.T。(2010)。 幼仓鼠肾(BHK)细胞的生长和维持 Curr Protoc 微生物第4章:附录4H
  2. Hernandez,R.,Sinodis,C.and Brown,D.T。(2005)。 Sindbis病毒:传播,量化和存储。 Curr Protoc Microbiol 第15章:单位15B 11。
  3. Piper,A.,Ribeiro,M.,Smith,KM,Briggs,CM,Huitt,E.,Nanda,K.,Spears,CJ,Quiles,M.,Cullen,J.,Thomas,ME,Brown, Hernandez,R。(2013)。 基孔肯雅病毒宿主范围E2跨膜缺失突变体诱导针对C57BL/6J小鼠中攻击的保护性免疫。 a> J Virol 87(12):6748-6757。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Briggs, C. M., Piper, A. and Hernandez, R. (2014). Chikungunya ELISA Protocol. Bio-protocol 4(4): e1049. DOI: 10.21769/BioProtoc.1049.
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